Resonance Energy Transfer

David L. Andrews and David S. Bradshaw

University of East Anglia

Abstract

Resonance energy transfer* is a spectroscopic process whose relevance in all

major areas of science is reflected both by a wide prevalence of the effect, and

through numerous technical applications. It is an optical near-field mechanism

which effects a transportation of electronic excitation between physically

distinct atomic or molecular components, based on transition dipole-dipole

coupling. In this chapter a comprehensive survey of the process is presented,

beginning with an outline of the history and highlighting the early contributions

of Perrin and Förster. A review of the photophysics behind resonance energy

transfer follows, and then a discussion of some prominent applications of

resonance energy transfer. Particular emphasis is given to techniques used in

molecular biology, ranging from the ‘spectroscopic ruler’ measurements of

functional group separation, to fluorescence lifetime microscopy. Finally,

applications to synthetic polymers and chemical sensors are examined.

1. History of RET

1.1 The first experiments

Resonance energy transfer (RET) is a process by means of which the energy of

an excited atom or molecule (usually called the donor, but known historically

as the ‘sensitizer’) is transferred non-radiatively to an acceptor molecule

(‘activator’), through intermolecular dipole-dipole coupling. The origins of its

discovery can be traced back to 1922, when the phenomenon of resonance

energy transfer (‘sensitized fluorescence’) was first experimentally observed by

Cario and Franck1-3 in the gas phase. This spectroscopic experiment involved

illuminating a mixture of mercury and thallium vapours at a wavelength

absorbed solely by the mercury; the resulting fluorescence spectra proving to

include frequencies that could only be emitted from thallium. Such energy

transfer in vapours was at first assumed to be uniquely associated with

inter-atomic collisions, but a discovery that transfer could occur at larger

separations than the collision radii showed that this was not necessarily the

case. Soon RET was also being observed in solutions,4 and over the following

years in many other physical systems.

* RET is also known as Förster- or fluorescence- resonance energy transfer (FRET), or electronic

energy transfer (EET)

1.2 Early developments of theory

The first theoretical explanation of the phenomenon was proposed by the Nobel

laureate J. Perrin.5 He recognized that energy could be transferred from an

excited molecule to its neighbours amongst closely spaced molecules through

dipole interactions; he named this process “transfert d’activation”, and his

paper on the subject became the earliest attempt to describe non-radiative

(near-field) energy transfer. Despite its initial success, however, Perrin’s

model incorrectly predicted that non-radiative energy transfer should be

possible between dye molecules up to an intermolecular distance of 1000 Å,

deriving from an inaccurate assumption that the molecules would act as

Hertzian oscillators with exactly defined resonance frequencies. Five years

later,6 Perrin’s son Francis developed a corresponding quantum mechanical

theory of RET, based on Kallman and London’s results.7 In this work he

recognized a “spreading of absorption and emission frequency” due to the

interactions of the dye with the solvent, thus reducing the probability of energy

transfer. As a result, efficient transfer was calculated to occur up to 150 - 250

Å, still approximately a factor of 3 greater than experimentally observed. A

detailed and readable survey of these early contributions of J. and F. Perrin can

be found in a review by Berberan-Santos.8

1.3 Förster theory

Extending the ideas of J. and F. Perrin, Förster developed the first essentially

correct theoretical treatment of RET.9-11 Förster determined that energy

transfer, through dipolar coupling between molecules, mostly depends on two

important quantities: spectrum overlap and intermolecular distance. Following

the observation that “the absorption and fluorescence spectra of similar

molecules are far from completely overlapping”, he found a means to quantify

the spectral overlap integral. The dipole-dipole interaction was known to have

an inverse proportionality on the cube of the molecular separation. Since the

rate of energy transfer is proportional to the square of this coupling, it thus

depends on the sixth power of the separation – i.e. the famous R-6 distance-

dependence law. Moreover, the acceptor distance at which this rate equates to

that of spontaneous emission by the donor, now termed the Förster radius R0,

was now calculated to be between 10 and 100 Å, in agreement with

experimental observations.

Much later, the distance dependence predicted by Förster was fully

verified by fluorescence studies of donor-acceptor pairs at known

separations,12,13 leading to the suggested employment of RET as a

‘spectroscopic ruler’ by Stryer and Haugland;13 hence, a technique to measure

the proximity relationships and conformational change in macromolecules was

realized (see Section 3). With the advent of the laser, in the 1960s, the modern

understanding of RET led to a raft of modern applications. An excellent

in-depth review on the history of RET is given by Clegg.14

2. The photophysics of RET

Resonance energy transfer is a mechanism that is now known to operate across

a diverse and extensive range of physical systems, encompassing not only

gases and dye solutions, but also protein complexes, doped crystals and

polymers, to name but a few. Nonetheless, at a fundamental level it is possible

to identify numerous common features in the underlying photophysics.

2.1 Primary excitation processes

To approach the subject in detail, let us commence with the photoexcitation

process that creates the conditions for RET to occur. When resonant ultraviolet

or visible radiation impinges on any non-homogeneous dielectric material, the

primary result of photon absorption is the population of electronic excited

states in individual atomic, molecular or other nanoscale centres – henceforth,

the latter are to be grouped together under the generic term ‘chromophore’.

Typically, such absorption is immediately followed by a rapid but partial

degradation of the acquired energy, the associated losses (largely due to

vibrational dissipation) ultimately to be manifest in the form of heat. This

effect owes its origin to the principle that the release of electronic energy by

fluorescence generally occurs from the lowest vibrational level of the excited

state. However, if any nearby chromophore has a suitably disposed electronic

state, of a similar or slightly lower energy, that neighbour may acquire the

major part of the electronic excitation through RET – a process that takes place

well before any further thermal degradation of the excited state energy occurs.

The mechanism is most commonly studied through spectrometric

differentiation of fluorescence emerging from the initially excited energy donor

and from the energy acceptor species, as illustrated in Fig. 1. As will be shown

in the following, the propensity for energy to be transferred between any two

chromophores is severely restricted by distance, and if no suitable acceptor is

within reach, the donor will generally shed its energy by fluorescence or local

dissipation.

2.2 Coupling of electronic transitions

In systems where resonance energy transfer occurs, the donors and acceptors

are usually fluorophores, i.e. chromophores that have the capacity to exhibit

fluorescence decay. Moreover, in RET, the transitions of donor decay and

acceptor excitation are generally electric dipole-allowed – although other

possibilities occasionally arise. Accordingly the theory of energy transfer, for

donor-acceptor displacements beyond the region of significant wavefunction

overlap, is traditionally conceived in terms of an electrodynamical coupling

between transition dipoles.

Consider the pairwise transfer of excitation between two chromophores

D and A. In the context of this elementary mechanism, D is designated the

donor and A the acceptor. Specifically, let it be assumed that prior excitation of

the donor generates an electronically excited species D*. Forward progress of

the energy is then accompanied by donor decay to the ground electronic state.

Acquiring the energy, D undergoes a transition from its ground to its excited

state. The complete RET process is generally a singlet-singlet coupling

mechanism†, due to the constraints of spin conservation (although this does not

apply to Dexter exchange, vide infra), and its entirety may be expressed by the

following chemical equation:

RET

1 * 1 1 1 *D A D A . (1)

The excited acceptor, A*, subsequently decays either in a further transfer event,

or by another means such as fluorescence. Since the D* and A* excited states

are real, with measurable lifetimes, the core process of energy transfer itself is

fundamentally separable from the initial electronic excitation of D and the

eventual decay of A; the latter processes do not, therefore, enter into the theory

of the pair transfer.

2.3 Dissipation and line-broadening

To delve more deeply into the nature of the process, it needs to be recognized

that equation (1) tells only part of the story, dealing as it does with only

electronic excitations. In general other, dissipative processes are also engaged.

In a solid, the linewidth of optical transitions manifests the influence of local

electronic environments which, in the case of strong coupling, may lead to the

production of phonon side-bands. Similar effects in solutions or disordered

solids represent inhomogeneous interactions with a solvent or host, while the

broad bands exhibited by chromophores in complex molecular systems signify

extensively overlapped vibrational levels, including those associated with

skeletal modes of the superstructure. In each case, energy level broadening can

allow pair transfer to occur at any point within the region of overlap between

the donor emission and acceptor absorption bands, as illustrated in Fig. 2.

2.4 The Förster equation

The Förster theory delivers an expression for the rate of pairwise energy

transfer, wF, valid for any donor-acceptor separation, R, that is substantially

smaller than the wavelengths of visible radiation. For systems where the

common host material for the donor and acceptor has refractive index n, at the

optical frequency corresponding to the mean transferred energy, the Förster

result is as follows:15

† Triplet-triplet energy transfer is also allowed by the Förster mechanism.

2 4

F 4 6 4

*

9 d( ) ( )

8D A

D

cw F

n R

. (2)

In this expression, FD() denotes the normalized fluorescence spectrum of the

donor, A() represents the linear absorption cross-section of the acceptor, and

is an optical frequency in radians per unit time; the specific form of the

integral within which they appear is known as the spectral overlap – one of the

key determinants of energy transfer efficiency.16 Also in equation (2), c is the

speed of light and D* is the associated radiative decay lifetime in the absence

of transfer. The latter is related to the measured fluorescence lifetime fl

through the fluorescence quantum yield fl *D , where 1

fl is explicitly

expressible as;

6

0

fl * *

1 1 1

D D

R

R

. (3)

The last term on the right-hand side of equation (3) is expressed with reference

to the Förster radius R0 – the distance at which the rates of donor deactivation

by RET and by spontaneous fluorescence become equal. As is evident from

Fig. 2, the propensity for forward transfer is usually significantly greater than

that for backward transfer, due to a sizeable difference in the spectral overlaps

for the two processes.

2.5 Orientation dependence

The factor in equation (2) depends on the orientations of the donor and

acceptor, both with respect to each other, and with respect to their mutual

displacement unit vector R̂ , as follows:

ˆ ˆˆ ˆ ˆ ˆ( ) 3( )( )D A D A μ μ R μ R μ . (4)

For each chromophore, μ̂ designates a unit vector in the direction of the

appropriate transition dipole moment. The possible values of 2, as featured in

equation (2), lie in the range (0, 4). It is evident that in the case of fixed

chromophore positions and orientations the result delivered by (4) is a function

of three independent angles, as shown and defined in Fig. 3:

cos 3cos cosT D A . (5)

Unfavorable orientations can thus reduce the rate of energy transfer to zero;

other configurations, including many of those found in photobiological

systems, optimize the transfer rate. The angular disposition of chromophores is

therefore a very important facet of energy transfer. It is important to note that

transfer is not necessarily precluded when the transition moments lie in

perpendicular directions – provided that neither is also disposed orthogonally to

R (= R R̂ ).

In any, at least partially, fluid or disordered system the relative

orientation of all donor-acceptor pairs may not be identical, and it is then the

distributional average of 2 that determines the overall measured response. In

the isotropic case (completely uncorrelated orientations) the 2 factor averages

to 23 ; departures from this value provide the quantitative signature of a degree

of orientational correlation. In molecules of sufficiently high symmetry it can

also happen that either the donor or the acceptor transition moment is not

unambiguously identifiable with a particular direction in the corresponding

chromophore reference frame. Specifically, the electronic transition may then

relate to a transition involving a degenerate state – as can occur with square

planar complexes, for example.17 Alternatively, the same observational

features might indicate rapid but orientationally confined motions. The

considerable complication which each of these effects brings into the

trigonometric analysis of RET has been extensively researched and reported by

van der Meer.18

2.6 Förster radius

In applications of RET to spectroscopy, as noted above, it is usually significant

that the electronically excited donor can in principle release its energy by

spontaneous decay, the ensuing fluorescence also being amenable to detection

by any suitably placed photodetector. Since the alternative possibility (that of

energy being transferred to another chromophore within the system) has such a

sharp decline in efficiency as the distance to the acceptor increases, it is

commonplace to invoke the critical distance R0. The Förster rate equation is

itself often cast in an alternative form, exactly equivalent to equation (2),

explicitly exhibiting this critical distance:19

62

0F

*

3 1

2 D

Rw

R

. (6)

Here the symbol with its over-bar, 0R , is defined as the Förster radius for

which the orientation factor 2 would assume its isotropic average value, 23 .20

For complex systems the angular dependence is quite commonly disregarded

and the following, simpler expression employed;

6

0F

*

1

D

Rw

R

, (7)

leading to a transfer efficiency T expressible as:

fl fl

Τ 6

0

11 1

1 D D

I

IR R

, (8)

where, flI and D

I are the intensities of the donor fluorescence with the

acceptor present and excluded, respectively, and fl specifically denotes the

fluorescence lifetime of the donor measured within its RET environment. As

graphically depicted in Fig. 4, a donor-acceptor displacement equal to R0

corresponds to a transfer efficiency of 50%.

The final equality on the right of equation (8), which holds provided

decay processes follow single-exponential decay kinetics, provides a formula

cast in terms of easily measurable quantities. This is particularly useful since it

allows energy transfer efficiencies to be calculated simply on the basis of

intensity measurements (for example using a fluorimeter), obviating the

separate time-resolved measurements that are otherwise generally necessary for

evaluation of the characteristic decay lifetimes fl and D*. When a given

electronically excited chromophore is within a distance R0 of a suitable

acceptor, RET will generally be the dominant decay mechanism; conversely,

for distances beyond R0, spontaneous decay (usually fluorescence) will be the

primary means of donor deactivation.

2.7 Polarization features

When linearly polarised laser light is used to excite any specific species within

a complex disordered solid or liquid system, the probability for excitation of

any particular molecule is proportional to cos2, where is the angle between

the appropriate excitation transition moment and the electric polarisation vector

of the input radiation. Consequently the population of excited molecules has a

markedly anisotropic distribution, a phenomenon associated with the term

photoselection. If radiative decay were to ensue instantaneously, i.e. from

precisely the initially populated excited level, then the fluorescence would

carry the full imprint of that anisotropy and itself exhibit a degree of

polarization – the highest value possible. Accounting for the necessary three-

dimensional rotational average,21 it is readily shown that the fluorescence

intensity components polarized parallel to and perpendicular to the polarization

of the excitation beam, I and I respectively, would then lie in the ratio 3:1.

Commonly observed departures from this result thus signify the extent to

which the orientation of the emission dipole differs from that of the prior,

initial excitation – which may be due to intervening decay, molecular motion or

intermolecular energy transfer.

The two most widely used quantitative expressions of polarization

retention are the fluorescence anisotropy, r, or the degree of polarization, P.

Both convey the same information; they are defined and related as follows:

rI I

I IP

I I

I Ir

P

P

2

2

3, . (9)

The denominator of the expression for r designates the net fluorescence

intensity. In a specific situation where the donor and acceptor have transition

dipole moments oriented in parallel, then r = 0.4 and P = 0.5.

A key molecular factor determining any loss in polarization is the angle

between the directions of the absorption and emission transition dipole

moments. In terms of this parameter and its influence on the measured

fluorescence anisotropy, the case where internal decay intervenes between

excitation and fluorescence decay within a single molecule is no different from

that of a donor-acceptor pair where the absorption and emission processes are

spatially separated – provided the donor and acceptor in the latter case have a

fixed mutual orientation (the orientation of the pair being random). The

following result, derived by Levshin22 and Perrin23 can be applied in both

situations:

2

2

cos3

1cos3

P . (10)

In the case of a donor-acceptor pair, is to be interpreted as the angle T shown

in Fig. 3. Equation (10) thus allows direct calculation of this microscopic

parameter, through measurement of the macroscopic quantity P. Moreover

when P proves to exhibit a time-dependent decay, a study of the kinetics

provides information on the extent of rotational motion intervening between the

absorption and emission events.

Very different behaviour is observed for RET systems in which the

donor and acceptor are orientationally uncorrelated, i.e. where they are both,

independently, randomly oriented. In such cases there is a very rapid loss of

polarization ‘memory’, and it transpires that the associated degree of

anisotropy is precisely 1/25, i.e. r = 0.04;24 two or more energy transfer jumps

will therefore usually, to all intents and purposes, totally destroy any

polarization in any ensuing fluorescence. However it should be noted that

there is a surprising recovery in the anisotropy at distances approaching the

transfer wavelength. The effect is sufficiently strong to warrant attention in

dilute solution studies.

2.8 Diffusion effects

So far only RET between a donor-acceptor pair has been considered. The

discussion is now extended to an ensemble of donors D and acceptors A, all

units of which are distributed randomly within an n-dimensional volume. For

systems in which translational diffusion is extremely slow compared to the rate

of energy transfer, the time-dependence of the donor intensity decay at time t,

*DI t , as obtained by Förster,11 is given by the following expression:

6

* *

* *

0 exp 2

n

D D

D D

t tI t I

. (11)

The most commonly applied form of this expression is when n equals 3, i.e.

RET in three dimensions. In equation (11), the parameter is explicitly

written as;

3

320

2

3AC R , (12)

in which CA is the concentration of acceptors (number per unit volume) and

3

04 3 AR C represents the average number of acceptor chromophores in a

sphere of radius 0R ; the orientational factor is again set as 23 .

The case where diffusion is comparable to the transfer rate is very

complicated, and calculations by Butler and Pilling25 have shown that large

errors arise on using Förster theory for systems with diffusion coefficients in

excess of 10–5 cm2 s–1. To address such systems, a successful approximation

was developed by Gösele et al.26 This approach involves the insertion of a

multiplier G within the second term in the exponential of equation (11). With

n = 3, the parameter G is given by;

3

2 41 5.47 4.00

1 3.34

x xG

x

, (13)

in which 1 3

6 2 3

0 *Dx D R t

, where D is the mutual diffusion coefficient. In

contrast to the Förster theory, the above method provides an excellent

approximation – as was fully verified by the authors of ref. 25.

2.9 Long-range transfer

Förster theory is found to be increasingly inaccurate for RET as donor-acceptor

distances extend over and beyond 100 Å. It was originally assumed that a

‘radiative’ process accounted for energy transfer over such donor-acceptor

separations, and some recent literature on the subject still perpetuate this initial

over-statement. Certain sources wrongly treat Förster ‘radiationless’ energy

transfer as exact, distinct and separable from ‘radiative’ energy transfer – the

latter signifying successive but independent processes of fluorescence emission

by a donor, and capture of the ensuing photon by an acceptor.

Although that certainly is the observed character of resonance energy

transfer over very long distances – as for example between donor and acceptor

components in a dilute solution – it is now known that both ‘radiative’ and

Förster transfer are simply the long- and short-range limits of one powerful, all-

pervasive mechanism. The latter, determined from quantum electrodynamical

calculations, is the outcome of the unified theory of RET.27 This theory not

only embraces Förster and ‘radiative’ energy transfer, but also addresses the

intermediate range in which neither of these mechanisms are fully valid. An

expression for the total pairwise energy transfer rate, ranging from molecular

dimensions up to interstellar distances, is written as;

F I radw w w w , (14)

where Fw represents the Förster rate of equation (2), radw is the rate of

‘radiative’ energy transfer – explicitly given by;

2

rad D A2

D

9( ) ( )d

8w F

R

, (15)

and Iw is the intermediate term that is expressed as:

2

2

I D A2 4 2

D

9( 2 ) ( ) ( )

8

c dw F

n R

. (16)

In both equation (15) and (16), the symbol denotes an orientation factor

identical to (4) but with the ‘3’ omitted from the second term. In summary, the

unified theory of RET contains not only the R-6 term of Förster theory and the

R-2 term denoting the inverse-square law of ‘radiative’ transfer, but also a

previously unidentified R-4 intermediate term.

2.10 Dexter transfer

Before concluding this section, it is worth observing that other forms of donor-

acceptor coupling are also possible, although considerably less relevant to the

systems of interest in the following focused account on applications. For

example, the transfer of energy between atomic or molecular components with

significantly overlapped wavefunctions is usually described in terms of Dexter

theory28 – where the coupling involves electron exchange and carries an

exponential decay with distance, directly reflecting the radial form of

overlapping wavefunctions and electron distributions. Unlike Förster transfer,

singlet-triplet energy exchange ( 3 * 1 1 3 *D A D A ) may also be allowed by

the Dexter mechanism. This is because Dexter transfer does not involve

transition dipole moments and, thus, is unaffected by the dipole-forbidden

character of the transitions 1 0T S and

0 1S T within chromophores D and A,

respectively. Compared to materials in which the donor and acceptor orbitals

do not spatially overlap, such systems are of less use for either device or

analytical applications. This is largely because the coupled chromophores lose

their electronic and optical integrity, the Dexter mechanism being operational

only at very short distances (< 10 Å).

3. Applications of RET to molecular biology

The field in which measurements of resonance energy transfer have without

doubt had the greatest impact is molecular biology. The importance of RET to

this subject, especially in application to biological macromolecules, was first

realised following the construction of spectroscopic equipment for routine

fluorescence measurements.29-32 Towards the turn of the twenty-first century,

RET underwent a period of significant redevelopment as a spectroscopic

technique.33 This resurgence arose mainly due to the advent of new

experimentation methods, for example single-pair RET,34 and further advances

in instrumentation. The key advantage of RET techniques over others is that

fluorescence measurements are highly sensitive, being made against a zero

background; moreover the uv/visible signals are relatively easy to detect, they

are specific and the required instrumentation is non-invasive.

3.1 Spectroscopic ruler

A major use of RET, based on its strong distance dependence, exploits its

capacity to supply accurate spatial information about molecular structures.

This derives from a quantitative assessment of the inter-chromophore

separations, based on comparisons between the corresponding RET

efficiencies.35-38 Such a technique is popularly known as a ‘spectroscopic

ruler’. The elucidations of molecular structure by such means usually lack

information on the relative orientations of the groups involved, and as an

expedient the calculations usually ignore the kappa parameter (4). The

apparent crudeness of this approach becomes more defensible on realizing that,

even if it were to introduce a factor of two inaccuracy, the deduced group

spacing would still be in error by only 12% (since 21/6 = 1.12). Refinements to

the theory to accommodate the effect of fluctuations in position or orientation

of the participant groups introduce considerable complexity, although progress

is being made in several areas.39-43

3.2 Conformational change

Through identification of motions in macromolecules, i.e. the variation in

proximity of one chromophore with respect to another, a number of valuable

RET applications arise; including the detection of conformational changes and

folding in proteins,36,44-46 and the inspection of intracellular protein-protein47-50

and protein-DNA51,52 interactions (see for example Figs 5 and 6). These and

other such processes can be registered by selectively exciting one chromophore

using laser light, and monitoring either the decrease in fluorescence from that

chromophore, or the rise in the generally longer-wavelength fluorescence from

the other chromophore as it adopts the role of acceptor. The judicious use of

optical dichroic filters can make this RET technique perfectly straightforward –

see Fig. 1. In cases where the two material components of interest do not

display suitably overlapped absorption and fluorescence features in an optically

accessible wavelength range, molecular tagging with site-specific ‘extrinsic’

(i.e. artificially attached) chromophores can solve the problem. Located at a

molecular site of interest, and being selected on the basis of a significant

spectral overlap with the counterpart component, such tags can act either in the

capacity of donor or acceptor. Lanthanide ions, with their characteristically

prominent and line-like absorption features, prove particularly valuable in this

connection.53 Also useful in this respect are the semiconductor nanocrystals

known as quantum dots. These crystalline nanoparticles offer several unique

traits, including size- and composition-tunable emission from visible to infrared

wavelengths, the possibility of a single light source simultaneous exciting

different-sized dots, large absorption coefficients across a wide spectral range,

and very high level of photostability.54,55

3.3 Intensity-based imaging

In the last two decades there has been burgeoning interest in microscopy based

on RET,56-60 typical instrumentation for which is illustrated in Fig. 7. There are

three specific types of RET method routinely used in the production of

biological images. The principles of sensitized-emission RET have already

been described (Fig. 1). For microscopy purposes this method is fairly

inaccurate; no RET donor-acceptor pair is ideal, i.e. there will almost always be

some overlap between the donor and acceptor absorbance bands and also the

donor and acceptor emission spectra. Therefore, filters that completely

separate these kinds of spectrum are difficult to design. Various calculational

algorithms61-63 have been proposed to compensate for this problem, although

the methods are complex and no single procedure has received universal

acceptance.

A widely used alternative, experimental approach64,65 involves

deliberately photobleaching the acceptor, the result of which is complete

exclusion of RET. In this method, the donor emission is analysed before and

after the acceptor is bleached by the input of an intense laser beam (at a

suitable wavelength). The difference between the donor intensities, with and

without the laser input, enables a determination of the transfer efficiency by

employing equation (8). Here, account is taken of spectral bleed-through

between the two absorbance bands, and equally between the two emission

bands. Signal contamination is still not entirely eliminated, due to a small

amount of back-transfer through donor excitation by acceptor emission. Often

the main disadvantage in prolonged illumination of the acceptor is the

possibility of damage to the sample. Therefore, in practice, photobleaching is

seldom appropriate for in vivo studies.

3.4 Lifetime-based imaging

Fluorescence need not be characterized from excitation and emission spectra

alone; highly significant information can also be secured from lifetime

measurements. Thus, when suitable time-resolved instrumentation is available,

the determination of decay kinetics (usually on the nanosecond timescale)

enables analysis through RET-based fluorescence lifetime imaging microscopy

(FRET-FLIM).66-69 In this method, spectral bleed-through is no longer an issue

since measurements are made only for the determination of donor lifetimes;

back-transfer is usually extremely low and within the noise level. The presence

of the acceptor within the local environment of the donor influences the

fluorescence lifetime of the donor. By measuring the donor lifetime in the

presence and absence of the acceptor one can accurately calculate the transfer

efficiency by use of equation (8). Drawbacks to FRET-FLIM are the technical

challenges the technique presents, and the expense of the equipment.

Nonetheless, in optical systems that are equipped to provide both intensity and

lifetime measurements, a comparison of the two types of image affords a

particularly rich source of information, as illustrated by the cancer cell images

of Fig. 8.

3.5 Other applications

Beyond the realm of molecular biology, RET has value in a number of more

specifically chemical applications. Two prominent examples are to be found in

the fields of synthetic macromolecules and chemical sensors. In polymer

science, building on the pioneering principles of Morawetz,70 RET is now used

to determine morphological information on polymer interfaces. Such studies

have, for instance, enabled the quantitative characterisation of interfacial

thickness in polymers of various structures.71 Moreover, RET has been utilized

in the study of polymer conformational dynamics. One especially interesting

application is the effective differentiation between various collapsed and/or

ordered homopolymer chain conformations (spherical, rod and toroidal; as

depicted in Fig. 9) through the associated distribution of transfer effiencies.72,73

The fabrication of RET-based, analyte-specific sensors has enabled

detection of a variety of species, including dimers of functionalized calixarenes

in organic solutions,74 copper(II) in aqueous solution,75 hydrogen peroxide,76

phosgene77 and many others. These chemical sensors usually work on the

principle of a donor-acceptor system designed such that the presence of the

analyte causes the acceptor chromophore to move within closer proximity to a

donor, enabling the implementation of an RET process that is not observed in

the analyte’s absence. Therefore, on irradiation of the system with the relevant

chemical present, a strong emission from the acceptor signals the presence of

the analyte.

Acknowledgements

We gratefully acknowledge helpful comments from Professor Steve Meech.

Research on the theory of RET, at the University of East Anglia, is currently

supported by the Leverhulme Trust.

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Further reading

D. L. Andrews and A. A. Demidov (eds), “Resonance Energy Transfer”,

(Wiley, New York, NY, USA, 1999).

D. L. Andrews, “Mechanistic principles and applications of resonance energy

transfer”, Canad. J. Chem., 86, pp. 855-870 (2008).

B. W. van der Meer, G. Coker and S.-Y. Chen, “Resonance Energy Transfer

Theory and Data”, (VCH, New York, 1994).

A. Periasamy, “Methods in cellular imaging”, (Oxford University Press, New

York, 2001).

A. Periasamy and R. N. Day, “Molecular imaging: FRET microscopy and

spectroscopy”, (Oxford University Press, New York, 2005).

K. E. Sapsford, L. Berti and I. L. Medintz, “Materials for fluorescence

resonance energy transfer analysis: Beyond traditional donor–acceptor

combinations”, Angew. Chem. Int. Ed. 45, pp. 4562- 4588 (2006).

G. D. Scholes, “Long-range resonance energy transfer in molecular systems”,

Annu. Rev. Phys. Chem. 54, pp. 57-87 (2003).

Review of FRET microscopy on Olympus Corporation’s website:

http://www.olympusfluoview.com/applications/fretintro.html (accessed July

2008).

Figure 1. Typical spectral discrimination between the fluorescence from donor and acceptor species

(here notionally based on a cyan fluorescent protein donor and a yellow fluorescent protein acceptor):

(a) the transmission characteristics of a short-wavelength filter ensure initial excitation of only the

donor; a dichroic beam-splitter and another narrow emission filter ensuring that only the (Stokes-

shifted) fluorescence from the donor reaches a detector; (b) in the same system a longer-wavelength

emission filter ensures capture of only the acceptor fluorescence, following RET.

Wavelength ()

Filt

er

Tra

nsm

issio

n (

%)

donor

absorption

donor

fluorescence

400 550500 600 650450

60

40

80

100

20

50

30

70

90

10

emission filter

excitation filter

dichroic

beamsplitter

Wavelength ()

acceptor

fluorescence

emission filter

excitation filter

dichroic

beamsplitter

400 550500 600 650450

60

40

80

100

20

50

30

70

90

10

donor

absorption

(a) (b)

Figure 2. Energetics and spectral overlap features (top) for energy transfer from D to A (and below,

potentially backward transfer from A to D). For each chromophore F denotes the fluorescence spectrum

and the absorption. Wavy downward lines denote vibrational dissipation.

R

D

A

A

D

T

Figure 3. Relative orientations and positions of the donor and acceptor and their transition moments:

Here, angles D and A subtended by donor and acceptor transition moments (D and A, respectively)

against the inter-chromophore displacement vector, R; the symbol T is the angle between the transition

moments.

Figure 4. Distance dependence of the transfer efficiency between a pair of chromophores, calculated

according to equation (8).

Figure 5. Graphical depiction of RET detection of protein conformational change. Adapted from

Olympus Corporation website.

Figure 6. Graphical depiction of RET detection of in vivo protein-protein interactions. Purple arrow

denotes input laser of wavelength 380 nm and green arrow indicates protein emission at 510 nm. BFP

and GFP are acronyms for blue and green fluorescent proteins, respectively. Adapted from Olympus

Corporation website.

Figure 7. Typical commercial set-up of a microscope based on RET. Adapted from Olympus

Corporation website.

Figure 8. MDA-MB-231 cancer cell images recorded with argon laser two-photon excitation, RET

microscope based on; (a) intensity and (b) fluorescence lifetime (ns). In the latter image, areas of

locally reduced lifetime signify clustered intracellular vesicles. Adapted from ref. 69.

Figure 9. Snapshots of various morphological constructions of a homopolymer chain. The structures

from top to bottom are spherical, rod and toroidal. Adapted from ref. 72.